Mammary transforming protein

ABSTRACT

A human mammary transforming protein and DNA (RNA) encoding such polypeptide and a procedure for producing such polypeptide by recombinant techniques is disclosed. Also disclosed are methods for inhibiting such polypeptide for preventing and/or treating neoplasia. Diagnostic assays for identifying mutations in nucleic acid sequence encoding a polypeptide of the present invention and for detecting altered levels of the polypeptide of the present invention for detecting diseases, for example, cancer, are also disclosed.

This application is entitled to the benefits of 35 U.S.C. § 1196 basedon U.S. Provisional Application Ser. No. 60/006,187, filed Nov. 2, 1995,pending.

This invention relates to newly identified polynucleotides, polypeptidesencoded by such polynucleotides, the use of such polynucleotides andpolypeptides, as well as the production of such polynucleotides andpolypeptides. More particularly, the polypeptide of the presentinvention has been putatively identified as mammary transformingprotein. The invention also relates to inhibiting the action of suchpolypeptides.

Hormones from ovaries and pituitary glands are absolutely essential forthe proliferation and differentiation of mammary epithelial cells(MECs), which are the predominant carcinogen-susceptible cell type inthe mammary gland (Imagawa, W., Bandyopadhyay, G. K. & Nandi, S. (1990)Endocr. Rev. 11, 494-523). Studies from several laboratories haveindicated that hormones play a crucial role in chemicalcarcinogen-induced mammary tumorigenesis in both mouse and rat modelsystems (Medina, D. (1974) J. Natl. Cancer Inst. 53, 223-226; Medina, D.(1976) J. Nat. Cancer Inst. 57, 1185-1189; Medina, D. (1981) Cancer Res.41, 3819-3820; Welsch, C. W. (1987) in Cellular and Molecular Biology ofMammary Cancer, eds. Medina, D., Kidwell, W. Heppner, G. & Anderson, E.(Plenum, New York), pp. 163-179). Earlier studies from differentlaboratories have demonstrated that the nature of the carcinogen and ofthe tissue types determine the genotype of the lesions induced usingvarious animal model systems. For example, in the two-stage skincarcinogenesis system, papillomas induced with the methylating agentN-methyl-N'-nitro-N-nitrosoguanidine or N-methyl-N-nitrosourea (MNU)have predominantly G→A transition mutations at codon 12 of the H-rasprotooncogene (Balmain, A. & Brown, K. (1988) adv. Cancer Res. 51,147-182; Brown, K., Buchmann, A. & Balmain, A. (1990) Proc. Natl. Acad.Sci. USA 87, 538-542). Similar findings have been reported in the ratmammary tumorigenesis system using MNU as a carcinogen (Sukumar, S.Notario, V., Martin-Zanca, D. & Barbacid, M. (1983) Nature (London) 306,658-661; Zarbl, H., Sukumar, S., Arthur, A. V., Martin-Sanca, D. &Barbacid, M. (1985) Nature (London) 315, 382-385). However, skin tumorsin mice and mammary tumors in mice and rats, induced with the polycyclichydrocarbon dimethylbenz [a] anthracene, contain predominantly A→Ttransversion mutations at the 61st codon of the H-ras protooncogene(Zarbl, H., Sukumar, S., Arthur, A. V., Martin-Sanca, D. & Barbacid, M.(1985) Nature (London) 315, 382-385; Kumar, R., MEdina, D. & Sukumar, S.(1990) Oncogene 5, 1271-1277; Dandekar, S., Sukumar, S., Zarbl, H.,Young, L. J. T. & Cardiff, R. D. (1986) Mol. Cell. Biol. 6,4104-4108;Quintanilla, M., Brown, K., Ramsden, M. & Balmain, A. (1986) Nature(London) 322, 78-80). A majority of thymic lymphomas induced with MNU,on the other hand, contain a G35→A35 mutation in the N-ras protooncogene(Guerrero, I., Calzada, P., Mayer, A. & Pellicer, A. (1984) Proc. Natl.Acad. Sci. USA 81, 202-205; Guerrero, I., Villasante, A., Corces, V. &Pellicer, A. (1985) Proc. Natl. Acad. Sci. USA 82, 7810-7814).

A defined serum-free cell culture system has been developed in whichmouse MECs embedded in a three-dimensional collagen gel matrix can begrown, induced to differentiate, and be neoplastically transformed withchemical carcinogens (Guzman, R. C., Osborn, R. C., Bartley, J. C.,Imagawa, W., Asch, B. B. & Nandi, S. (1987) Cancer Res. 47, 275-280).Using this system it has been observed that the types of mammary lesionsinduced by carcinogens are greatly influenced by the mitogens presentaround the time of carcinogen treatment. It has been reported on an invitro system, the induction of preneoplastic hyperplastic alveolarnodules (HANs) and carcinomas from MECs exposed to the direct-actingchemical carcinogen MNU in the presence of different mitogens (Miyamoto,S., Guzman, R. C., Osborn, R. C. & Nandi, S. (1988) Proc. Natl. Acad.Sci. USA 85, 477-481). When mouse MECs were grown in the presence of themammogenic hormones progestone and prolactin (PPRL) during MNUadministration, the predominant types of lesions induced were a highincidence of HANs and carcinomas with squamous metaplasia. In contrast,when epidermal growth factor was used as a mitogen during the carcinogentreatment, only a low incidence of ductal hyperplasia was detected,although the extent of MEC proliferation between the two groups wasequivalent. The genetic analysis of these lesions indicated that theactivation of the protooncogene was also dependent on the mitogen usedaround the time of carcinogen treatment. The majority (80%) of the HANsand carcinomas induced with MNU in the presence of PPRL had anactivation of the protooncogene c-Ki-ras by a specific G35→A35 pointmutation at codon 12. The activation of the protooncogene was determinedto be an early event in this carcinogenesis process because theactivation was detected in preneoplastic lesions (Miyamoto, S., Sukumar,S., Guzman, R. C., Osborn, R. C. & Nandi, S. (1990) Mol. Cell. Biol. 10,1593-1599). In contrast, activation of C-Ki-ras was absent in all theductal hyperplasias induced by MNU in the presence of the mitogenepidermal growth factor. Involvement of the same type of c-Ki-rasmutation has, however, been observed in the in vivo mouse model systemwhere pituitary-isografted mice were injected with a single dose of MNU(Guzman, R. C., Osborn, R. C., Swanson, S. M., Sakthivel, R., Hwang,S.-i., Miyamoto, S. & Nandi, S. (1992) Cancer Res. 52, 5732-5737).Pituitary isografts in mice raise blood levels of PPRL (Christov, K.,Swanson, S. M., Guzman, R. C., Thordarson, G., Jin, E., Talamantes, F. &Nandi, S. (1993) Carcinogenesis, 14, 2019-2025) and thereby partiallymimic the in vitro PPRL culture condition. Results from another set ofin vivo experiments with virgin rats also showed that a difference inexperiments with virgin rats also showed that a difference in frequencyof G35→A35 mutated H-ras protooncogene correlated with different stagesof the estrous cycle at the time of MNU administration (Pascual, R. V.,Hwang, S.-I., Swanson, S. M., Bauzon, M. K., Guzman, R. C. & Nandi, S.(1994) Proc. Am. Assoc. Cancer Res. 35, 262).

The induction of preneoplastic and neoplastic lesions of differentphenotypes by using LiCl as a mitogen during carcinogen treatment andthe involvement of a transforming gene, designated MAT1, in thisprocess, LiCl, a potent mitogen for mammary epithelial cells, has beenreported, (Hori, C. & Oka, T. (1979) Proc. Natl. Acad. Sci. USA 76,2823-2827; Tomooka, Y., Imagawa, W., Nandi, S. & Bern, H. A. (1983) J.Cell. Physiol. 117, 290-296). LiCl has been found to alter thephosphatidylinositol hydrolysis in MECs. Although LiCl also modules thecAMP synthesis, K⁺ and Ca²⁺ transport, and guanine nucleotide-bindingprotein synthesis in other cell types, the exact mechanism of itsmitogenic effect is still unclear (Imagawa, W., Bandyopadhyay, G. K. &Nandi, S. Endocr. Rev. 11:494-523 (1990)). This gene has been cloned andsequenced.

The polypeptide of the present invention has been putatively identifiedas a mammary transforming protein as a result of amino acid sequencehomology to mammary transforming gene (MAT1) as disclosed in Bera, T.,et al., PNAS, USA, 91:9789-9793 (1994).

In accordance with one aspect of the present invention, there isprovided a novel mature polypeptide, as well as biologically active anddiagnostically or therapeutically useful fragments, analogs andderivatives thereof. The polypeptide of the present invention is ofhuman origin.

In accordance with another aspect of the present invention, there areprovided isolated nucleic acid molecules encoding a polypeptide of thepresent invention including mRNAs, cDNAs, genomic DNAs as well asanalogs and biologically active and diagnostically or therapeuticallyuseful fragments thereof.

In accordance with another aspect of the present invention there isprovided an isolated nucleic acid molecule encoding a mature polypeptideexpressed by the DNA contained in ATCC Deposit No. 97300.

In accordance with yet a further aspect of the present invention, thereis provided a process for producing such polypeptide by recombinanttechniques comprising culturing recombinant prokaryotic and/oreukaryotic host cells, containing a nucleic acid sequence encoding apolypeptide of the present invention, under conditions promotingexpression of said protein and subsequent recovery of said protein.

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing such polypeptide, or polynucleotideencoding such polypeptide for therapeutic purposes, for example, toregulate development and normal physiology of cells.

In accordance with yet a further aspect of the present invention, thereare provided antibodies against such polypeptides.

In accordance with yet another aspect of the present invention, thereare provided antagonists to such polypeptides, which may be used toinhibit the action of such polypeptides, for example, to prevent thetransformation of cells which lead to neoplasia.

In accordance with yet a further aspect of the present invention, thereis also provided nucleic acid probes comprising nucleic acid moleculesof sufficient length to specifically hybridize to a nucleic acidsequence of the present invention.

In accordance with still another aspect of the present invention, thereare provided diagnostic assays for detecting diseases or susceptibilityto diseases related to an overexpression of a polypeptide of the presentinvention.

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing such polypeptides, orpolynucleotides encoding such polypeptides, for in vitro purposesrelated to scientific research, for example, synthesis of DNA andmanufacture of DNA vectors.

These and other aspects of the present invention should be apparent tothose skilled in the art from the teachings herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are illustrative of embodiments of the inventionand are not meant to limit the scope of the invention as encompassed bythe claims.

FIG. 1 is an illustration of the cDNA (SEQ ID NO:1) and correspondingdeduced amino acid sequence (SEQ ID NO:2) of the polypeptide of thepresent invention. Sequencing was performed using a 373 automated DNAsequencer (Applied Biosystems, Inc.).

FIG. 2 is an amino acid sequence comparison between the polypeptide ofthe present invention (top line) and mouse mammary transforming proteinas disclosed in Bera, et al., supra, (bottom line) (SEQ ID NO:9).

In accordance with an aspect of the present invention, there is providedan isolated nucleic acid (polynucleotide) which encodes for the maturepolypeptide having the deduced amino acid sequence of FIG. 1 (SEQ IDNO:2).

A polynucleotide encoding a polypeptide of the present invention may beobtained from a cDNA library derived from a human hypothalamus. It ismost closely related to the mammary transforming gene MAT1. It containsan open reading frame encoding a protein of 74 amino acid residues. Theprotein exhibits the highest degree of homology at the amino acid levelto the mouse mammary transforming gene MAT1 with 63.934% identity and73.770% similarity over the entire amino acid stretch, and at the aminoacid level the polynucleotide of the present invention exhibits 98%identity and 98% similarity to the human homolog of mouse MAT1 gene overa 120 nucleotide stretch. The polypeptide of the present invention has amolecular weight of 8445.10 daltons, has a length of 74 amino acids, amolar extinction coefficient of 849 and an isoelectric point of 10.02.

In accordance with another aspect of the present invention there areprovided isolated polynucleotides encoding a mature polypeptideexpressed by the DNA contained in ATCC Deposit No. 97300, deposited withthe American Type Culture Collection, 10801 University Boulevard,Manassas, Va. 20110-2209, USA, on September 25, 1995. The depositedmaterial is a plasmid that contains the full-length MTP CDNA insertedinto a pBluescript SK(-) vector (Stratagene, La Jolla, Calif.).

The deposit has been made under the terms of the Budapest Treaty on theInternational Recognition of the Deposit of Micro-organisms for purposesof Patent Procedure. The strain will be irrevocably and withoutrestriction or condition released to the public upon the issuance of apatent. The deposit is provided merely as convenience to those of skillin the art and are not an admission that a deposit is required under 35U.S.C. §112. The sequence of the polynucleotide contained in thedeposited material, as well as the amino acid sequence of thepolypeptides encoded thereby, are controlling in the event of anyconflict with any description of sequences herein. A license may berequired to make, use or sell the deposited material, and no suchlicense is hereby granted.

The polynucleotide of the present invention may be in the form of RNA orin the form of DNA, which DNA includes cDNA, genomic DNA, and syntheticDNA. The DNA may be double-stranded or single-stranded, and if singlestranded may be the coding strand or non-coding (anti-sense) strand. Thecoding sequence which encodes the mature polypeptide may be identical tothe coding sequence shown in FIG. 1 (SEQ ID NO:1), as a result of theredundancy or degeneracy of the genetic code, encodes the same maturepolypeptide as the DNA of FIG. 1 (SEQ ID NO:1).

The polynucleotide which encodes for the mature polypeptide of FIG. 1(SEQ ID NO:2) may include, but is not limited to: only the codingsequence for the mature polypeptide; the coding sequence for the maturepolypeptide and additional coding sequence such as a leader or secretorysequence or a proprotein sequence; the coding sequence for the maturepolypeptide (and optionally additional coding sequence) and non-codingsequence, such as introns or non-coding sequence 5' and/or 3' of thecoding sequence for the mature polypeptide.

Thus, the term "polynucleotide" encompasses a polynucleotide whichincludes only coding sequence for the polypeptide as well as apolynucleotide which includes additional coding and/or non-codingsequence.

The present invention further relates to variants of the hereinabovedescribed polynucleotides which encode for fragments, analogs andderivatives of the polypeptide having the deduced amino acid sequence ofFIG. 1 (SEQ ID NO:2). The variant of the polynucleotide may be anaturally occurring allelic variant of the polynucleotide or anon-naturally occurring variant of the polynucleotide.

Thus, the present invention includes polynucleotides encoding the samemature polypeptide as shown in FIG. 1 (SEQ ID NO:2) as well as variantsof such polynucleotides which variants encode for a fragment, derivativeor analog of the polypeptide of FIG. 1 (SEQ ID NO:2). Such nucleotidevariants include deletion variants, substitution variants and additionor insertion variants.

As hereinabove indicated, the polynucleotide may have a coding sequencewhich is a naturally occurring allelic variant of the coding sequenceshown in FIG. 1 (SEQ ID NO:1). As known in the art, an allelic variantis an alternate form of a polynucleotide sequence which may have asubstitution, deletion or addition of one or more nucleotides, whichdoes not substantially alter the function of the encoded polypeptide.

The present invention also includes polynucleotides, wherein the codingsequence for the mature polypeptide may be fused in the same readingframe to a polynucleotide sequence which aids in expression andsecretion of a polypeptide from a host cell, for example, a leadersequence which functions as a secretory sequence for controllingtransport of a polypeptide from the cell. The polypeptide having aleader sequence is a preprotein and may have the leader sequence cleavedby the host cell to form the mature form of the polypeptide. Thepolynucleotides may also encode for a proprotein which is the matureprotein plus additional 5' amino acid residues. A mature protein havinga prosequence is a proprotein and is an inactive form of the protein.Once the prosequence is cleaved an active mature protein remains.

Thus, for example, the polynucleotide of the present invention mayencode for a mature protein, or for a protein having a prosequence orfor a protein having both a prosequence and a presequence (leadersequence).

The polynucleotides of the present invention may also have the codingsequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence may be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or, for example, the marker sequencemay be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

The term "gene" means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

Fragments of the full length gene of the present invention may be usedas a hybridization probe for a cDNA library to isolate the full lengthcDNA and to isolate other cDNAs which have a high sequence similarity tothe gene or similar biological activity. Probes of this type preferablyhave at least 30 bases and may contain, for example, 50 or more bases.The probe may also be used to identify a cDNA clone corresponding to afull length transcript and a genomic clone or clones that contain thecomplete gene including regulatory and promotor regions, exons, andintrons. An example of a screen comprises isolating the coding region ofthe gene by using the known DNA sequence to synthesize anoligonucleotide probe. Labeled oligonucleotides having a sequencecomplementary to that of the gene of the present invention are used toscreen a library of human cDNA, genomic DNA or mRNA to determine whichmembers of the library the probe hybridizes to.

The present invention further relates to polynucleotides which hybridizeto the hereinabove-described sequences if there is at least 70%,preferably at least 90%, and more preferably at least 95% identitybetween the sequences. The present invention particularly relates topolynucleotides which hybridize under stringent conditions to thehereinabove-described polynucleotides. As herein used, the term"stringent conditions" means hybridization will occur only if there isat least 95% and preferably at least 97% identity between the sequences.The polynucleotides which hybridize to the hereinabove describedpolynucleotides in a preferred embodiment encode polypeptides whicheither retain substantially the same biological function or activity asthe mature polypeptide encoded by the cDNAs of FIG. 1 (SEQ ID NO:1).

Alternatively, the polynucleotide may have at least 20 bases, preferably30 bases, and more preferably at least 50 bases which hybridize to apolynucleotide of the present invention and which has an identitythereto, as hereinabove described, and which may or may not retainactivity. For example, such polynucleotides may be employed as probesfor the polynucleotide of SEQ ID NO:1, for example, for recovery of thepolynucleotide or as a diagnostic probe or as a PCR primer.

Thus, the present invention is directed to polynucleotides having atleast a 70% identity, preferably at least 90% and more preferably atleast a 95% identity to a polynucleotide which encodes the polypeptideof SEQ ID NO:2 and polynucleotides complementary thereto as well asportions thereof, which portions have at least 30 consecutive bases andpreferably at least 50 consecutive bases and to polypeptides encoded bysuch polynucleotides.

The present invention further relates to a polypeptide which has thededuced amino acid sequence of FIG. 1 (SEQ ID NO:2), as well asfragments, analogs and derivatives of such polypeptide.

The terms "fragment," "derivative" and "analog" when referring to thepolypeptide of FIG. 1 (SEQ ID NO:2), means a polypeptide which retainsessentially the same biological function or activity as suchpolypeptide. Thus, an analog includes a proprotein which can beactivated by cleavage of the proprotein portion to produce an activemature polypeptide.

The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide,preferably a recombinant polypeptide.

The fragment, derivative or analog of the polypeptide of FIG. 1 (SEQ IDNO:2) may be (i) one in which one or more of the amino acid residues aresubstituted with a conserved or non-conserved amino acid residue(preferably a conserved amino acid residue) and such substituted aminoacid residue may or may not be one encoded by the genetic code, or (ii)one in which one or more of the amino acid residues includes asubstituent group, or (iii) one in which the mature polypeptide is fusedwith another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol), or (iv) one in whichthe additional amino acids are fused to the mature polypeptide, such asa leader or secretory sequence or a sequence which is employed forpurification of the mature polypeptide or a proprotein sequence. Suchfragments, derivatives and analogs are deemed to be within the scope ofthose skilled in the art from the teachings herein.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The term "isolated" means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polypeptides of the present invention include the polypeptide of SEQID NO:2 (in particular the mature polypeptide) as well as polypeptideswhich have at least 80% similarity (preferably at least 80% identity) tothe polypeptide of SEQ ID NO:2 and more preferably at least 90%similarity (more preferably at least 90% identity) to the polypeptide ofSEQ ID NO:2 and still more preferably at least 95% similarity (stillmore preferably at least 95% identity) to the polypeptide of SEQ ID NO:2and also include portions of such polypeptides with such portion of thepolypeptide generally containing at least 30 amino acids and morepreferably at least 50 amino acids.

As known in the art "similarity" between two polypeptides is determinedby comparing the amino acid sequence and its conserved amino acidsubstitutes of one polypeptide to the sequence of a second polypeptide.

Fragments or portions of the polypeptides of the present invention maybe employed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, the fragments may be employed asintermediates for producing the full-length polypeptides. Fragments orportions of the polynucleotides of the present invention may be used tosynthesize full-length polynucleotides of the present invention.

The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed ortransfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the genes of the present invention. Theculture conditions, such as temperature, pH and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct mRNAsynthesis. As representative examples of such promoters, there may bementioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or moreselectable marker genes to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein.

As representative examples of appropriate hosts, there may be mentioned:bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium;fungal cells, such as yeast; insect cells such as Drosophila S2 andSpodoptera Sf 9; animal cells such as CHO, COS or Bowes melanoma;adenoviruses; plant cells, etc. The selection of an appropriate host isdeemed to be within the scope of those skilled in the art from theteachings herein.

More particularly, the present invention also includes recombinantconstructs comprising one or more of the sequences as broadly describedabove. The constructs comprise a vector, such as a plasmid or viralvector, into which a sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example; Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pBS, pD10, phagescript, psiX174, pBluescript SK, pBSKS, pNH8A, pNH16a,pNH18A, pNH46A (Stratagene); pTRC99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia); Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene)pSVK3, PBPV, PMSG, PSVL (Pharmacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cellscontaining the above-described constructs. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (Davis, L., Dibner, M., Battey, I.,Basic Methods in Molecular Biology, (1986)).

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), thedisclosure of which is hereby incorporated by reference.

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples include the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

Useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

As a representative but nonlimiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis.,USA). These pBR322 "backbone" sections are combined with an appropriatepromoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell known to those skilled in the art.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman,Cell, 23:175 (1981), and other cell lines capable of expressing acompatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5' flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

The polypeptide can be recovered and purified from recombinant cellcultures by methods including ammonium sulfate or ethanol precipitation,acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Protein refolding steps can be used, as necessary, incompleting configuration of the mature protein. Finally, highperformance liquid chromatography (HPLC) can be employed for finalpurification steps.

The polypeptides of the present invention may be a naturally purifiedproduct, or a product of chemical synthetic procedures, or produced byrecombinant techniques from a prokaryotic or eukaryotic host (forexample, by bacterial, yeast, higher plant, insect and mammalian cellsin culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay also include an initial methionine amino acid residue.

The polypeptide of the present invention plays a role in normaldevelopment and in normal physiological functions and may be employed insuch a manner to induce the appropriate biological effect in a host.

The polynucleotides and polypeptides of the present invention may beemployed as research reagents and materials for discovery of treatmentsand diagnostics to human disease.

This invention provides a method for identification of the receptor forthe mammary transforming protein. The gene encoding the receptor can beidentified by numerous methods known to those of skill in the art, forexample, ligand panning and FACS sorting (Coligan, et al., CurrentProtocols in Immun., 1(2), Chapter 5, (1991)). Preferably, expressioncloning is employed wherein polyadenylated RNA is prepared from a cellresponsive to the mammary transforming protein, and a cDNA librarycreated from this RNA is divided into pools and used to transfect COScells or other cells that are not responsive to the mammary transformingprotein. Transfected cells which are grown on glass slides are exposedto labeled mammary transforming protein. The mammary transformingprotein can be labeled by a variety of means including iodination orinclusion of a recognition site for a site-specific protein kinase.Following fixation and incubation, the slides are subjected toauto-radiographic analysis. Positive pools are identified and sub-poolsare prepared and re-transfected using an iterative sub-pooling andre-screening process, eventually yielding a single clone that encodesthe putative receptor. As an alternative approach for receptoridentification, labeled ligand can be photoaffinity linked with cellmembrane or extract preparations that express the receptor molecule.Cross-linked material is resolved by PAGE and exposed to X-ray film. Thelabeled complex containing the ligand-receptor can be excised, resolvedinto peptide fragments, and subjected to protein microsequencing. Theamino acid sequence obtained from microsequencing would be used todesign a set of degenerate oligonucleotide probes to screen a cDNAlibrary to identify the gene encoding the putative receptor.

The present invention also provides an assay to determine the activityof the protein of the present invention. The assay will determine thegrowth promoting activity of mammary transforming protein in vitro by aserum-free cell culture system in which mouse primary mammary epithelialcells can grow and differentiate in response to specific mammogenichormones and related growth factors. This serum-free culture system mayalso be employed to test the growth-promoting activity of mammarytransforming protein singly and in combination with other growth factorson mammary epithelial cells. The mutant mammary transforming protein maybe used in this assay to determine the effect of wild-type mammarytransforming protein onthe growth of mammary epithelial cells.

An in vivo assay to test the effect of the mammary transforming proteinof the present invention on the growth and morphogenesis of mammaryepithelial cells may be tested both in in-tact and ovariectomized mice.The protein is administered into the in-tact and ovariectomized mice andthe growth-promoting activity will be determined by Brd Uptake as wellas by whole mount preparation. The mammary transforming protein isadministered into the animal by several ways: (1) direct injection intothe target tissue; (2) mammary transforming protein pellet is made andimplanted into the animal and the mammary transforming protein isadministered by osmotic pump. The mammary transforming proteingrowth-promoting activity on human cells in vivo may also be tested bytransplanting the collagen embedded human cells into the athymic nudemice. For a review, see Bera, T., et al. PNAS, USA, 91:9789-9798 (1994).

This invention provides a method of screening compounds to identifythose which block interaction of mammary transforming protein with itsreceptor. As an example, a mammalian cell or membrane preparationexpressing the mammary transforming protein receptor would be incubatedwith labeled ligand in the presence of the drug. The ability of the drugto block this interaction could then be measured. Alternatively, theresponse of a known second messenger system following interaction ofligand and receptor would be measured and compared in the presence orabsence of the drug. Such second messenger systems include but are notlimited to, cAMP guanylate cyclase, ion channels or phosphoinositidehydrolysis. Another example of an assay combines mammary transformingprotein and a potential antagonist with membrane-bound receptors orrecombinant receptors under appropriate conditions for a competitiveinhibition assay. Mammary transforming protein can be labeled, such asby radioactivity, such that the number of molecules bound to thereceptor can determine the effectiveness of the potential antagonist.

Potential antagonists include an antibody, or in some cases, anoligopeptide, which binds to the polypeptide. Alternatively, a potentialantagonist may be a closely related protein which binds to the receptorsites, however, they are inactive forms of the polypeptide and therebyprevent the action of mammary transforming protein since receptor sitesare occupied.

Another potential antagonist is an antisense construct prepared usingantisense technology. Antisense technology can be used to control geneexpression through triple-helix formation or antisense DNA or RNA, bothof which methods are based on binding of a polynucleotide to DNA or RNA.For example, the 5' coding portion of the polynucleotide sequence, whichencodes for the mature polypeptides of the present invention, is used todesign an antisense RNA oligonucleotide of from about 10 to 40 basepairs in length. A DNA oligonucleotide is designed to be complementaryto a region of the gene involved in transcription (triple helix--see Leeet al., Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456(1988); and Dervan et al., Science, 251: 1360 (1991)), therebypreventing transcription and the production of mammary transformingprotein. The antisense RNA oligonucleotide hybridizes to the mRNA invivo and blocks translation of the mRNA molecule into mammarytransforming protein polypeptide (Antisense--Okano, J. Neurochem.,56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, Fla. (1988)). The oligonucleotidesdescribed above can also be delivered to cells such that the antisenseRNA or DNA may be expressed in vivo to inhibit production of mammarytransforming protein.

Potential antagonists include a small molecule which binds to andoccupies the catalytic site of the polypeptide thereby making thecatalytic site inaccessible to substrate such that normal biologicalactivity is prevented. Examples of small molecules include but are notlimited to small peptides or peptide-like molecules.

The antagonists may be employed to prevent the mammary transformingprotein of the present invention from neoplastically transforming cells.The antagonists may be employed in a composition with a pharmaceuticallyacceptable carrier, e.g., as hereinafter described.

The polypeptides of the present invention may be employed in combinationwith a suitable pharmaceutical carrier. Such compositions comprise atherapeutically effective amount of the polypeptide, and apharmaceutically acceptable carrier or excipient. Such a carrierincludes but is not limited to saline, buffered saline, dextrose, water,glycerol, ethanol, and combinations thereof. The formulation should suitthe mode of administration.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, thepolypeptides of the present invention may be employed in conjunctionwith other therapeutic compounds.

The pharmaceutical compositions may be administered in a convenientmanner such as by the oral, topical, parenterally, intravenous,intraperitoneal, intramuscular, subcutaneous, intranasal or intradermalroutes. The pharmaceutical compositions are administered in an amountwhich is effective for treating and/or prophylaxis of the specificindication. In general, they are administered in an amount of at leastabout 10 μg/kg body weight and in most cases they will be administeredin an amount not in excess of about 8 mg/Kg body weight per day. In mostcases, the dosage is from about 10 μg/kg to about 1 mg/kg body weightdaily, taking into account the routes of administration, symptoms, etc.

The mammary transforming protein polypeptides and agonists andantagonists which are polypeptides may also be employed in accordancewith the present invention by expression of such polypeptides in vivo,which is often referred to as "gene therapy."

Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with theengineered cells then being provided to a patient to be treated with thepolypeptide. Such methods are well-known in the art and are apparentfrom the teachings herein. For example, cells may be engineered by theuse of a retroviral plasmid vector containing RNA encoding a polypeptideof the present invention.

Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by, for example, procedures known in the art. Forexample, a packaging cell is transduced with a retroviral plasmid vectorcontaining RNA encoding a polypeptide of the present invention such thatthe packaging cell now produces infectious viral particles containingthe gene of interest. These producer cells may be administered to apatient for engineering cells in vivo and expression of the polypeptidein vivo. These and other methods for administering a polypeptide of thepresent invention by such method should be apparent to those skilled inthe art from the teachings of the present invention.

Retroviruses from which the retroviral plasmid vectors hereinabovementioned may be derived include, but are not limited to, Moloney MurineLeukemia Virus, spleen necrosis virus, retroviruses such as Rous SarcomaVirus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemiavirus, human immunodeficiency virus, adenovirus, MyeloproliferativeSarcoma Virus, and mammary tumor virus. In one embodiment, theretroviral plasmid vector is derived from Moloney Murine Leukemia Virus.

The vector includes one or more promoters. Suitable promoters which maybe employed include, but are not limited to, the retroviral LTR; theSV40 promoter; and the human cytomegalovirus (CMV) promoter described inMiller, et al., Biotechnicrues, Vol. 7, No. 9, 980-990 (1989), or anyother promoter (e.g., cellular promoters such as eukaryotic cellularpromoters including, but not limited to, the histone, pol III, andβ-actin promoters). Other viral promoters which may be employed include,but are not limited to, adenovirus promoters, thymidine kinase (TK)promoters, and B19 parvovirus promoters. The selection of a suitablepromoter will be apparent to those skilled in the art from the teachingscontained herein.

The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Suitablepromoters which may be employed include, but are not limited to,adenoviral promoters, such as the adenoviral major late promoter; orhetorologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRshereinabove described); the β-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe gene encoding the polypeptide.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, ψ2,ψ-AM, PA12, T19-14X, VT-19-17-H2, ψCRE, ψCRIP, GP+E-86, GP+envAm12, andDAN cell lines as described in Miller, Human Gene Therapy, Vol. 1, pgs.5-14 (1990), which is incorporated herein by reference in its entirety.The vector may transduce the packaging cells through any means known inthe art. Such means include, but are not limited to, electroporation,the use of liposomes, and CaPO₄ precipitation. In one alternative, theretroviral plasmid vector may be encapsulated into a liposome, orcoupled to a lipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particleswhich include the nucleic acid sequence(s) encoding the polypeptides.Such retroviral vector particles then may be employed, to transduceeukaryotic cells, either in vitro or in vivo. The transduced eukaryoticcells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

This invention is also related to the use of the gene of the presentinvention as a diagnostic. Detection of a mutated form of the gene willallow a diagnosis of a disease or a susceptibility to a disease whichresults from underexpression of mammary transforming protein.

Individuals carrying mutations in the gene of the present invention maybe detected at the DNA level by a variety of techniques. Nucleic acidsfor diagnosis may be obtained from a patient's cells, including but notlimited to blood, urine, saliva, tissue biopsy and autopsy material. Thegenomic DNA may be used directly for detection or may be amplifiedenzymatically by using PCR (Saiki et al., Nature, 324:163-166 (1986))prior to analysis. RNA or cDNA may also be used for the same purpose. Asan example, PCR primers complementary to the nucleic acid encodingmammary transforming protein can be used to identify and analyzemutations. For example, deletions and insertions can be detected by achange in size of the amplified product in comparison to the normalgenotype. Point mutations can be identified by hybridizing amplified DNAto radiolabeled RNA or alternatively, radiolabeled antisense DNAsequences. Perfectly matched sequences can be distinguished frommismatched duplexes by RNase A digestion or by differences in meltingtemperatures.

Sequence differences between the reference gene and genes havingmutations may be revealed by the direct DNA sequencing method. Inaddition, cloned DNA segments may be employed as probes to detectspecific DNA segments. The sensitivity of this method is greatlyenhanced when combined with PCR. For example, a sequencing primer isused with double-stranded PCR product or a single-stranded templatemolecule generated by a modified PCR. The sequence determination isperformed by conventional procedures with radiolabeled nucleotide or byautomatic sequencing procedures with fluorescent-tags.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230:1242 (1985)).

Sequence changes at specific locations may also be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401 (1985)).

Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,Restriction Fragment Length Polymorphisms (RFLP)) and Southern blottingof genomic DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations can also be detected by in situ analysis.

The present invention also relates to diagnostic assays for detectingthe presence or over-expression of the polypeptide of the presentinvention in host tissues, for example histological sections of mammarysections or in blood, since an over-expression of the proteins comparedto normal control tissue samples can detect the presence of neoplasia,for example, cancer. Assays used to detect levels of the polypeptide ofthe present invention in a sample derived from a host are well-known tothose of skill in the art and include radioimmunoassays,competitive-binding assays, Western Blot analysis and preferably anELISA assay. An ELISA assay initially comprises preparing an antibodyspecific to a mammary transforming protein antigen, preferably amonoclonal antibody. In addition a reporter antibody is prepared againstthe monoclonal antibody. To the reporter antibody is attached adetectable reagent such as radioactivity, fluorescence or in thisexample a horseradish peroxidase enzyme. A sample is now removed from ahost and incubated on a solid support, e.g. a polystyrene dish, thatbinds the proteins in the sample. Any free protein binding sites on thedish are then covered by incubating with a non-specific protein such asbovine serum albumin. Next, the monoclonal antibody is incubated in thedish during which time the monoclonal antibodies attached to any of thepolypeptide of the present invention attached to the polystyrene dish.All unbound monoclonal antibody is washed out with buffer. The reporterantibody linked to horseradish peroxidase is now placed in the dishresulting in binding of the reporter antibody to any monoclonal antibodybound to the polypeptide of the present invention. Unattached reporterantibody is then washed out. Peroxidase substrates are then added to thedish and the amount of color developed in a given time period is ameasurement of the amount of the polypeptide of the present inventionpresent in a given volume of patient sample when compared against astandard curve.

A competition assay may be employed wherein antibodies specific to thepolypeptide of the present invention are attached to a solid support andlabeled mammary transforming protein and a sample derived from the hostare passed over the solid support and the amount of label detectedattached to the solid support can be correlated to a quantity of thepolypeptide of the present invention in the sample. These assays mayalso be used to monitor cancer progression, remission and recurrence.

The sequences of the present invention are also valuable for chromosomeidentification. The sequence is specifically targeted to and canhybridize with a particular location on an individual human chromosome.Moreover, there is a current need for identifying particular sites onthe chromosome. Few chromosome marking reagents based on actual sequencedata (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp) from the cDNA. Computer analysis of the 3'untranslated region of the gene is used to rapidly select primers thatdo not span more than one exon in the genomic DNA, thus complicating theamplification process. These primers are then used for PCR screening ofsomatic cell hybrids containing individual human chromosomes. Only thosehybrids containing the human gene corresponding to the primer will yieldan amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of fragments from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphasechromosomal spread can be used to provide a precise chromosomal locationin one step. This technique can be used with cDNA having at least 50 or60 bases. For a review of this technique, see Verma et al., HumanChromosomes: a Manual of Basic Techniques, Pergamon Press, New York(1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man (available on line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

The polypeptides, their fragments or other derivatives, or analogsthereof, or cells expressing them can be used as an immunogen to produceantibodies thereto. These antibodies can be, for example, polyclonal ormonoclonal antibodies. The present invention also includes chimeric,single chain, and humanized antibodies, as well as Fab fragments, or theproduct of an Fab expression library. Various procedures known in theart may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler and Milstein, 1975,Nature, 256:495-497), the trioma technique, the human B-cell hybridomatechnique (Kozbor et al., 1983, Immunology Today 4:72), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole, etal., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic polypeptide products of this invention. Also, transgenicmice may be used to express humanized antibodies to immunogenicpolypeptide products of this invention.

The present invention will be further described with reference to thefollowing examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

In order to facilitate understanding of the following examples certainfrequently occurring methods and/or terms will be described.

"Plasmids" are designated by a lower case p preceded and/or followed bycapital letters and/or numbers. The starting plasmids herein are eithercommercially available, publicly available on an unrestricted basis, orcan be constructed from available plasmids in accord with publishedprocedures. In addition, equivalent plasmids to those described areknown in the art and will be apparent to the ordinarily skilled artisan.

"Digestion" of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fragments is performed using 8 percentpolyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res.,8:4057 (1980).

"Oligonucleotides" refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5' phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

"Ligation" refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (Maniatis, T. , et al., Id.,p. 146). Unless otherwise provided, ligation may be accomplished usingknown buffers and conditions with 10 units of T4 DNA ligase ("ligase")per 0.5 μg of approximately equimolar amounts of the DNA fragments to beligated.

Unless otherwise stated, transformation was performed as described inthe method of Graham, F. and Van der Eb, A., Virology, 52:456-457(1973).

EXAMPLE 1

Bacterial Expression and Purification of Mammary Transforming Protein

The DNA sequence encoding mammary transforming protein, ATCC # 97300, isinitially amplified using PCR oligonucleotide primers corresponding tothe 5' sequences of the processed mammary transforming protein (minusthe signal peptide sequence) and the vector sequences 3' to the mammarytransforming protein gene. Additional nucleotides corresponding tomammary transforming protein were added to the 5' and 3' sequencesrespectively. The 5' oligonucleotide primer has the sequence 5'CGCGGATCCGCCATCATGTATATTAAAACTGCA 3' (SEQ ID NO:3) contains a BamHIrestriction enzyme site followed by 18 nucleotides of the mammarytransforming protein coding sequence. The 3' sequence 5' CGCGGATCCCTAAAAGCTCCTAACTTG 3' (SEQ ID NO:4) contains complementary sequences toa BamHI site and is followed by 18 nucleotides of mammary transformingprotein including the stop codon. The restriction enzyme sitescorrespond to the restriction enzyme sites on the bacterial expressionvector pQE-9 (Qiagen, Inc. Chatsworth, Calif., 91311). pQE-9 encodesantibiotic resistance (Amp^(r)), a bacterial origin of replication(ori), an IPTG-regulatable promoter operator (P/O), a ribosome bindingsite (RBS), a 6-His tag and restriction enzyme sites. pQE-9 was thendigested with BamHI. The amplified sequences were ligated into pQE-9 andwere inserted in frame with the sequence encoding for the histidine tagand the RBS. The ligation mixture was then used to transform E. colistrain M15/rep 4 (Qiagen, Inc.) by the procedure described in Sambrook,J. et al., Molecular Cloning: A Laboratory Manual, Cold SpringLaboratory Press, (1989). M15/rep4 contains multiple copies of theplasmid pREP4, which expresses the lacI repressor and also conferskanamycin resistance (Kanr). Transformants are identified by theirability to grow on LB plates and ampicillin/kanamycin resistant colonieswere selected. Plasmid DNA was isolated and confirmed by restrictionanalysis. Clones containing the desired constructs were grown overnight(O/N) in liquid culture in LB media supplemented with both Amp (100ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a largeculture at a ratio of 1:100 to 1:250. The cells were grown to an opticaldensity 600 (O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG("Isopropyl-B-D-thiogalacto pyranoside") was then added to a finalconcentration of 1 mM. IPTG induces by inactivating the lacI repressor,clearing the P/O leading to increased gene expression. Cells were grownan extra 3 to 4 hours. Cells were then harvested by centrifugation. Thecell pellet was solubilized in the chaotropic agent 6 Molar GuanidineHCl. After clarification, solubilized mammary transforming protein waspurified from this solution by chromatography on a Nickel-Chelate columnunder conditions that allow for tight binding by proteins containing the6-His tag (Hochuli, E. et al., J. Chromatography 411:177-184 (1984)).Mammary transforming protein (90% pure) was eluted from the column in 6molar guanidine HCl pH 5.0 and for the purpose of renaturation adjustedto 3 molar guanidine HCl, 100 mM sodium phosphate, 10 mmolar glutathione(reduced) and 2 mmolar glutathione (oxidized). After incubation in thissolution for 12 hours the protein was dialyzed to 10 mmolar sodiumphosphate.

EXAMPLE 2

Cloning and expression of mammary transforming protein using thebaculovirus expression system

The DNA sequence encoding the full length mammary transforming protein,ATCC # 97300, was amplified using PCR oligonucleotide primerscorresponding to the 5' and 3' sequences of the gene:

The 5' primer has the sequence 5' CGCGGATCCGCCATCATGTAT ATTAAAACTGCA 3'(SEQ ID NO:5) and contains a BamHI restriction enzyme site (in bold)followed by 6 nucleotides resembling an efficient signal for theinitiation of translation in eukaryotic cells (Kozak, M., J. Mol. Biol.,196:947-950 (1987) which is just behind the first 18 nucleotides of themammary transforming protein gene (the initiation codon for translation"ATG" is underlined).

The 3' primer has the sequence 3' CGCGGATCCCTAAAAGCTCCT AACTTG 5' (SEQID NO:6) and contains the cleavage site for the restriction endonucleaseBamHI and 18 nucleotides complementary to the 3' translated sequence ofthe mammary transforming protein gene and stop codon. The amplifiedsequences were isolated from a 1% agarose gel using a commerciallyavailable kit ("Geneclean," BIO 101 Inc., La Jolla, Calif.). Thefragment was then digested with the endonuclease BamHI and then purifiedagain on a 1% agarose gel. This fragment is designated F2.

The vector pA2-Gp (modification of pVL941 vector, discussed below) isused for the expression of the mammary transforming protein using thebaculovirus expression system (for review see: Summers, M. D. and Smith,G. E. 1987, A manual of methods for baculovirus vectors and insect cellculture procedures, Texas Agricultural Experimental Station Bulletin No.1555). This expression vector contains the strong polyhedrin promoter ofthe Autographa californica nuclear polyhedrosis virus (AcMNPV) followedby the recognition sites for the restriction endonuclease BamHI. Thepolyadenylation site of the simian virus (SV)40 is used for efficientpolyadenylation. For an easy selection of recombinant virus thebeta-galactosidase gene from E.coli is inserted in the same orientationas the polyhedrin promoter followed by the polyadenylation signal of thepolyhedrin gene. The polyhedrin sequences are flanked at both sides byviral sequences for the cell-mediated homologous recombination ofco-transfected wild-type viral DNA. Many other baculovirus vectors couldbe used in place of pRG1 such as pAc373, pVL941 and pAcIM1 (Luckow, V.A. and Summers, M. D., Virology, 170:31-39).

The plasmid was digested with the restriction enzymes BamHI, thendephosphorylated using calf intestinal phosphatase by procedures knownin the art. The DNA was then isolated from a 1% agarose gel using thecommercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Calif.).This vector DNA is designated V2.

Fragment F2 and the dephosphorylated plasmid V2 were ligated with T4 DNAligase. E.coli HB101 cells were then transformed and bacteria identifiedthat contained the plasmid (pBac mammary transforming protein) with themammary transforming protein gene using the enzymes BamHI. The sequenceof the cloned fragment was confirmed by DNA sequencing.

5 μg of the plasmid pBac mammary transforming protein was co-transfectedwith 1.0 μg of a commercially available linearized baculovirus("BaculoGold™ baculovirus DNA", Pharmingen, San Diego, Calif.) using thelipofection method (Felgner et al. Proc. Natl. Acad. Sci. USA,84:7413-7417 (1987)).

1 μg of BaculoGold™ virus DNA and 5 μg of the plasmid pBac mammarytransforming protein were mixed in a sterile well of a microtiter platecontaining 50 μl of serum free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards 10 μl Lipofectin plus 90 μl Grace'smedium were added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture was added drop-wise to theSf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture platewith 1 ml Grace's medium without serum. The plate was rocked back andforth to mix the newly added solution. The plate was then incubated for5 hours at 27DC. After 5 hours the transfection solution was removedfrom the plate and 1 ml of Grace's insect medium supplemented with 10%fetal calf serum was added. The plate was put back into an incubator andcultivation continued at 27° C. for four days.

After four days the supernatant was collected and a plaque assayperformed similar as described by Summers and Smith (supra). As amodification an agarose gel with "Blue Gal" (Life Technologies Inc.,Gaithersburg) was used which allows an easy isolation of blue stainedplaques. (A detailed description of a "plaque assay" can also be foundin the user's guide for insect cell culture and baculovirologydistributed by Life Technologies Inc., Gaithersburg, page 9-10).

Four days after the serial dilution, the virus was added to the cellsand blue stained plaques were picked with the tip of an Eppendorfpipette. The agar containing the recombinant viruses was thenresuspended in an Eppendorf tube containing 200 μl of Grace's medium.The agar was removed by a brief centrifugation and the supernatantcontaining the recombinant baculovirus was used to infect Sf9 cellsseeded in 35 mm dishes. Four days later the supernatants of theseculture dishes were harvested and then stored at 4° C.

Sf9 cells were grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells were infected with the recombinantbaculovirus V-mammary transforming protein at a multiplicity ofinfection (MOI) of 2. Six hours later the medium was removed andreplaced with SF900 II medium minus methionine and cysteine (LifeTechnologies Inc., Gaithersburg). 42 hours later 5 μCi of ³⁵S-methionine and 5 μCi ³⁵ S cysteine (Amersham) were added. The cellswere further incubated for 16 hours before they were harvested bydialysis agains PBS and centrifugation and the labelled proteinsvisualized by SDS-PAGE and autoradiography.

EXAMPLE 3

Expression of Recombinant Mammary Transforming Protein in COS cells

The expression of plasmid, mammary transforming protein HA is derivedfrom a vector pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin ofreplication, 2) ampicillin resistance gene, 3) E.coli replicationorigin, 4) CMV promoter followed by a polylinker region, an SV40 intronand polyadenylation site. A DNA fragment encoding the entire mammarytransforming protein precursor and a HA tag fused in frame to its 3' endis cloned into the polylinker region of the vector, therefore, therecombinant protein expression is directed under the CMV promoter. TheHA tag corresponds to an epitope derived from the influenzahemagglutinin protein as previously described (I. Wilson, H. Niman, R.Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37:767,(1984)). The infusion of HA tag to the target protein allows easydetection of the recombinant protein with an antibody that recognizesthe HA epitope.

The plasmid construction strategy is described as follows:

The DNA sequence encoding mammary transforming protein, ATCC # 97300, isconstructed by PCR using two primers: the 5' primer 5'GCGCGGATCCACCATGTATATTAAACTGCA 3' (SEQ ID NO:7) contains a BamHI sitefollowed by 18 nucleotides of mammary transforming protein codingsequence starting from the initiation codon; the 3' sequence 5'GCGCTCTAGATCAAGCGTA GTCTGGGACGTCGTATGGGTAAAAGCTCCTAACTTG (SEQ ID NO:8)contains complementary sequences to an XbaI site, translation stopcodon, HA tag and the last 15 nucleotides of mammary transformingprotein coding sequence (not including the stop codon). Therefore, thePCR product contains a BamHI site, mammary transforming protein codingsequence followed by HA tag fused in frame, a translation terminationstop codon next to the HA tag, and an XbaI site. The PCR amplified DNAfragment and the vector, pcDNAI/Amp, are digested with BamHI and XbaIrestriction enzyme and ligated. The ligation mixture is transformed intoE. coli strain SURE (Stratagene Cloning Systems, La Jolla, Calif.) thetransformed culture is plated on ampicillin media plates and resistantcolonies are selected. Plasmid DNA is isolated from transformants andexamined by restriction analysis for the presence of the correctfragment. For expression of the recombinant mammary transformingprotein, COS cells are transfected with the expression vector byDEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis, MolecularCloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)). Theexpression of the mammary transforming protein HA protein is detected byradiolabelling and immunoprecipitation method (Harlow, E. and Lane, D.,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,(1988)). Cells are labelled for 8 hours with ³⁵ S-cysteine two days posttransfection. Culture media is then collected and cells are lysed withdetergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5%DOC, 50 mM Tris, pH 7.5) (Wilson, I. et al., Id. 37:767 (1984)). Bothcell lysate and culture media are precipitated with an HA specificmonoclonal antibody. Proteins precipitated are analyzed on 15% SDS-PAGEgels.

EXAMPLE 4

Expression via Gene Therapy

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in tissue-culture medium and separated into smallpieces. Small chunks of the tissue are placed on a wet surface of atissue culture flask, approximately ten pieces are placed in each flask.The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin, is added. This is then incubated at 37° C. forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerge. The monolayer istrypsinized and scaled into larger flasks.

pMV-7 (Kirschmeier, P. T. et al, DNA, 7:219-25 (1988) flanked by thelong terminal repeats of the Moloney murine sarcoma virus, is digestedwith EcoRI and HindIII and subsequently treated with calf intestinalphosphatase. The linear vector is fractionated on agarose gel andpurified, using glass beads.

The cDNA encoding a polypeptide of the present invention is amplifiedusing PCR primers which correspond to the 5' and 3' end sequencesrespectively. The 5' primer containing an EcoRI site and the 3' primerfurther includes a HindIII site. Equal quantities of the Moloney murinesarcoma virus linear backbone and the amplified EcoRI and HindIIIfragment are added together, in the presence of T4 DNA ligase. Theresulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is used to transformbacteria HB101, which are then plated onto agar-containing kanamycin forthe purpose of confirming that the vector had the gene of interestproperly inserted.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissueculture to confluent density in Dulbecco's Modified Eagles Medium (DMEM)with 10% calf serum (CS), penicillin and streptomycin. The MSV vectorcontaining the gene is then added to the media and the packaging cellsare transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently,the media is harvested from a 10 cm plate of confluent producer cells.The spent media, containing the infectious viral particles, is filteredthrough a millipore filter to remove detached producer cells and thismedia is then used to infect fibroblast cells. Media is removed from asub-confluent plate of fibroblasts and quickly replaced with the mediafrom the producer cells. This media is removed and replaced with freshmedia. If the titer of virus is high, then virtually all fibroblastswill be infected and no selection is required. If the titer is very low,then it is necessary to use a retroviral vector that has a selectablemarker, such as neo or his.

The engineered fibroblasts are then injected into the host, either aloneor after having been grown to confluence on cytodex 3 microcarrierbeads. The fibroblasts now produce the protein product.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                  - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES:  9                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  928 BAS - #E PAIRS                                               (B) TYPE:  NUCLEIC A - #CID                                                   (C) STRANDEDNESS:  SING - #LE                                                 (D) TOPOLOGY:  LINEAR                                                - -     (ii) MOLECULE TYPE:  cDNA                                             - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #1:                          - - TACTTAGGAA ACCCTTCTTT ACTAGATATC TGGCCCCCTG GGCTTGTGAA CA -             #CCTCCTAG     60                                                                 - - CCACATCACT ACAGTACAGT GAGTGACCCC AGCCTCCTGC CTACCCCAAG AT -            #GCCCCTCC    120                                                                 - - CCACCCTGAC CGTGCTAACT GTGTGTACAT ATATATTCTA CATAT ATG - #TAT ATT       AAA    177                                                                      Met Tyr Ile Lys                                                                - - ACT GCA CTG CCA TGT CTG CCC TTT TTT GTG GT - #G TCT AGC ATT AAC        TTA      225                                                                    Thr Ala Leu Pro Cys Leu Pro Phe Phe Val Va - #l Ser Ser Ile Asn Leu          5                  - # 10                 - # 15                 - # 20        - - TTG TCT AGG CCA GAG CGG GGG TGG GAG GGG AA - #T GCC ACA GTG AAG GGA          273                                                                       Leu Ser Arg Pro Glu Arg Gly Trp Glu Gly As - #n Ala Thr Val Lys Gly           25                  - #30                  - #35                               - - GTG GGC AGA ATC AAA TTG CTA CAT AGT CCA AA - #C AAA AAA GAG GCT TTT          321                                                                       Val Gly Arg Ile Lys Leu Leu His Ser Pro As - #n Lys Lys Glu Ala Phe           40                  - #45                  - #50                               - - TCA AAA AAC ATT AAA TTC ACA TGC AGT CTC AG - #A GAC TAT TTA GAC AAA          369                                                                       Ser Lys Asn Ile Lys Phe Thr Cys Ser Leu Ar - #g Asp Tyr Leu Asp Lys           55                  - #60                  - #65                               - - GTT CAA GTT AGG AGC TTT TAG GATGTGGGAG TAAAACTTT - #A ATGGGAGGGG             420                                                                       Val Gln Val Arg Ser Phe                                                       70                                                                             - - AGGGCTGGCT GCTGGAAGAA GGAAGAAGCC AGACTGGTTA GACAGTACTC TT -             #AACTCCTA    480                                                                 - - GCCCAGCCTA GCGTGCCCTG CCCCTCTGGC CACTGCTGCA GACACCTGCC TT -            #AACACACA    540                                                                 - - CACCTCTAGG ACTCCACAGT TTTGCCTTAA AGGACCTTCC CAAGTCTCCC TT -            #CCCCTGTC    600                                                                 - - TGGCTTCTCC TTTAAGAAGA GAGAGATACT TGTAGAATTG GGTGGGGGGA AT -            #GAGCATGA    660                                                                 - - ACTGTCCTTC CATTTGGGAT ATGTTACATT AGAGTGAGAG AGAGAATAAG GA -            #GCCTTTCT    720                                                                 - - TATGGAAGAA ATGGGAGAAG AGAGACAGGG TTCTTTTCAG CAGAGTCTAG TA -            #GTTTCTCT    780                                                                 - - GTAAGGCAAA ATAATCTAAA AAGACTAACC TGCCCACCCA CTCCTTATAT TG -            #CTGTGAGA    840                                                                 - - TTGCCCCTAT CTTGTGCTCT TCTGTCTGCA GTGTGCACGG CCTTGTTCTA AC -            #CCGGAATA    900                                                                 - - AAGGTGATTG ATTGTATTGG CAAAAAAA         - #                  - #                928                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  74 AMIN - #O ACIDS                                               (B) TYPE:  AMINO ACI - #D                                                     (C) STRANDEDNESS:                                                             (D) TOPOLOGY:  LINEAR                                                - -     (ii) MOLECULE TYPE:  PROTEIN                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #2:                          - - Met Tyr Ile Lys Thr Ala Leu Pro Cys Leu Pr - #o Phe Phe Val Val                            - #5                  - #10                  - #15           - - Ser Ser Ile Asn Leu Leu Ser Arg Pro Glu Ar - #g Gly Trp Glu Gly                           20 - #                 25 - #                 30              - - Asn Ala Thr Val Lys Gly Val Gly Arg Ile Ly - #s Leu Leu His Ser                           35 - #                 40 - #                 45              - - Pro Asn Lys Lys Glu Ala Phe Ser Lys Asn Il - #e Lys Phe Thr Cys                           50 - #                 55 - #                 60              - - Ser Leu Arg Asp Tyr Leu Asp Lys Val Gln Va - #l Arg Ser Phe                               65 - #                 70                                     - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  33 BASE - # PAIRS                                                (B) TYPE:  NUCLEIC A - #CID                                                   (C) STRANDEDNESS:  SING - #LE                                                 (D) TOPOLOGY:  LINEAR                                                - -     (ii) MOLECULE TYPE:  Oligonucleotide                                  - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #3:                          - - CGCGGATCCG CCATCATGTA TATTAAAACT GCA       - #                  - #             33                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  27 BASE - # PAIRS                                                (B) TYPE:  NUCLEIC A - #CID                                                   (C) STRANDEDNESS:  SING - #LE                                                 (D) TOPOLOGY:  LINEAR                                                - -     (ii) MOLECULE TYPE:  Oligonucleotide                                  - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #4:                          - - CGCGGATCCC TAAAAGCTCC TAACTTG          - #                  - #                 27                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  33 BASE - # PAIRS                                                (B) TYPE:  NUCLEIC A - #CID                                                   (C) STRANDEDNESS:  SING - #LE                                                 (D) TOPOLOGY:  LINEAR                                                - -     (ii) MOLECULE TYPE:  Oligonucleotide                                  - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #5:                          - - CGCGGATCCG CCATCATGTA TATTAAAACT GCA       - #                  - #             33                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  27 BASE - # PAIRS                                                (B) TYPE:  NUCLEIC A - #CID                                                   (C) STRANDEDNESS:  SING - #LE                                                 (D) TOPOLOGY:  LINEAR                                                - -     (ii) MOLECULE TYPE:  Oligonucleotide                                  - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #6:                          - - CGCGGATCCC TAAAAGCTCC TAACTTG          - #                  - #                 27                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  30 BASE - # PAIRS                                                (B) TYPE:  NUCLEIC A - #CID                                                   (C) STRANDEDNESS:  SING - #LE                                                 (D) TOPOLOGY:  LINEAR                                                - -     (ii) MOLECULE TYPE:  Oligonucleotide                                  - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #7:                          - - GCGCGGATCC ACCATGTATA TTAAACTGCA         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:   55 - #BASE PAIRS                                                (B) TYPE:  NUCLEIC A - #CID                                                   (C) STRANDEDNESS:  SING - #LE                                                 (D) TOPOLOGY:  LINEAR                                                - -     (ii) MOLECULE TYPE:  Oligonucleotide                                  - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #8:                          - - GCGCTCTAGA TCAAGCGTAG TCTGGGACGT CGTATGGGTA AAAGCTCCTA AC - #TTG              55                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  61 AMIN - #O ACIDS                                               (B) TYPE:  AMINO ACI - #D                                                     (C) STRANDEDNESS:                                                             (D) TOPOLOGY:  LINEAR                                                - -     (ii) MOLECULE TYPE:  PROTEIN                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #9:                          - - Met Tyr Ile Lys Pro Ala Leu Pro Cys Leu Pr - #o Phe Phe Val Val                            - #5                  - #10                  - #15           - - Phe Ser Ile Asn Leu Leu Ser Arg Pro Glu Ar - #g Glu Trp Glu Gly                           20 - #                 25 - #                 30              - - Met Pro Gln Lys Gly Ser Gly Arg Ala Lys Le - #u Leu Gln Ser Pro                           35 - #                 40 - #                 45              - - Asn Arg Lys His Ile Ser Thr Asn His Asn Ly - #s Cys Tyr Ile Tyr                           50 - #                 55 - #                 60              - - Met                                                                     __________________________________________________________________________

What is claimed is:
 1. An isolated polynucleotide comprising a nucleicacid sequence selected from the group consisting of:(a) a nucleic acidsequence encoding the polypeptide set forth as amino acid residues 1 to74 of SEQ ID NO:2; (b) a nucleic acid sequence encoding the polypeptideset forth as amino acid residues 2 to 74 of SEQ ID NO:2; (c) a nucleicacid sequence encoding a fragment of the polypeptide set forth as aminoacid residues 1 to 74 of SEQ ID NO:2 wherein said fragment retainscellular proliferation activity; and (d) a nucleic acid sequencecomplementary to the nucleic acid sequence of (a), (b) or (c).
 2. Theisolated polynucleotide of claim 1 wherein the nucleic acid sequence is(a).
 3. The isolated polynucleotide of claim 1 wherein the nucleic acidsequence is (b).
 4. The isolated polynucleotide of claim 1 wherein thenucleic acid sequence is (c).
 5. The isolated polynucleotide of claim 1wherein said polynucleotide is DNA and further wherein said nucleic acidsequence is (a), (b) or (c).
 6. A recombinant vector comprising the DNAof claim
 5. 7. A recombinant host cell comprising the DNA of claim
 5. 8.A polynucleotide comprising the DNA of claim 5 linked to a heterologousregulatory sequence which controls gene expression.
 9. A process forproducing a polypeptide comprising expressing from the host cell ofclaim 7 the encoded polypeptide and recovering said polypeptide.
 10. Theisolated polynucleotide of claim 1 consisting of a nucleic acid sequenceselected from the group consisting of:(a) a nucleic acid sequenceencoding the polypeptide set forth as amino acid residues 1 to 74 of SEQID NO:2; (b) a nucleic acid sequence encoding the polypeptide set forthas amino acid residues 2 to 74 of SEQ ID NO:2; (c) a nucleic acidsequence encoding a fragment of the polypeptide set forth as amino acidresidues 1 to 74 of SEQ ID NO:2 wherein said fragment retains cellularproliferation activity; and (d) a nucleic acid sequence complementary tothe nucleic acid sequence of (a), (b) or (c).
 11. An isolatedpolynucleotide comprising the polynucleotide of claim 10 fused to aheterologous polynucleotide.
 12. An isolated polynucleotide comprising anucleic acid sequence selected from the group consisting of:(a) anucleic acid sequence encoding the amino acid sequence of thepolypeptide encoded by the human cDNA contained in ATCC Deposit No.97300; (b) a nucleic acid sequence encoding the amino acid sequence of afragment of the polypeptide encoded by the human cDNA contained in ATCCDeposit No. 97300 wherein said fragment retains cellular proliferationactivity; (c) a nucleic acid sequence complementary to the nucleic acidsequence of (a) or (b).
 13. The isolated polynucleotide of claim 12wherein said nucleic acid sequence is (a).
 14. The isolatedpolynucleotide of claim 12 wherein said nucleic acid sequence is (b).15. The isolated polynucleotide of claim 12 wherein said polynucleotideis DNA and further wherein said nucleic acid sequence is (a) or (b). 16.A recombinant vector comprising the DNA of claim
 15. 17. A recombinanthost cell comprising the DNA of claim
 15. 18. The isolated DNA of claim15 linked to regulatory sequence which controls gene expression.
 19. Aprocess for producing a polypeptide comprising expressing from the hostcell of claim 17 the encoded polypeptide and recovering saidpolypeptide.
 20. The isolated polynucleotide of claim 12 consisting of anucleic acid sequence selected from the group consisting of:(a) anucleic acid sequence encoding the amino acid sequence of thepolypeptide encoded by the human CDNA contained in ATCC Deposit No.97300; (b) a nucleic acid sequence encoding the amino acid sequence of afragment of the polypeptide encoded by the human CDNA contained in ATCCDeposit No. 97300 wherein said fragment retains cellular proliferationactivity; (c) a nucleic acid sequence complementary to the nucleic acidsequence of (a) or (b).
 21. An isolated polynucleotide comprising thepolynucleotide of claim 20 fused to a heterologous polynucleotide.